Studies are proposed to investigate what host and phage proteins interact in order to link lambda DNA to the replication machinery of E. coli. The principal experimental approach will be to follow the fate of lambda DNA in E. coli lysogenic for lambda. The advantage of such a system is that the repression system can be made thermally labile by the presence of a temperature sensitive repressor; thus repression can be lifted or imposed by shifts in temperature. Within 2-3 minutes, superinfecting lambda DNA circles are nicked and associate with a rapidly sedimenting host complex (RSC) by a process that is under sole control of lambda gene N. Experiments are planned to investigate whether the RSC formation represents association of lambda DNA with the inner or outer membranes of E. coli, and if attachment sites for lambda DNA compete with attachment sites for the "folded chromosomes" of E. coli. Bacterial mutants will be sought which are attachment defective. Of particular interest will be mutants that have temperature sensitive components in DNA replication. These will be examined for defects in their ability to attach both the host "folded chromosome", as well as for RSC formation at temperatures at which the thermally labile component is inactive. A major effort is planned to examine the proteins of lambda DNA-membrane complexes by labeling them with S35 and subjecting them to SDS gel electrophoresis. Of particular interest will be the N protein. An investigation will be made to determine if N protein becomes associated with the inner or outer host membrane in the presence or absence of intact lambda DNA using bacterial strains that contain essentially only the N and cI857 genes of lambda. The presence of the latter gene results in production of thermally labile cI857 repressor. Thus, synthesis of N gene protein can be controlled by the temperature of the bacterial culture. At 42 degrees, in the presence of S35 the N protein will be tagged and accumulated within the cell in the absence or presence of infecting lambda DNA. Its identity will be confirmed by comparing results obtained from control cells lacking the N gene.